Mapping of the RNA recognition site of Escherichia coli ribosomal protein S7

Francis Robert, Matthieu Gagnon, Dimitri Sans, Stephen Michnick, Léa Brakier-Gingras

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

Bacterial ribosomal protein S7 initiates the folding of the 3' major domain of 16S ribosomal RNA by binding to its lower half. The X-ray structure of protein S7 from thermophilic bacteria was recently solved and found to be a modular structure, consisting of an α-helical domain with a β-ribbon extension. To gain further insights into its interaction with rRNA, we cloned the S7 gene from Eschsrichia coli K12 into a pET expression vector and introduced 4 deletions and 12 amino acid substitutions in the protein sequence. The binding of each mutant to the lower half of the 3' major domain of 16S rRNA was assessed by filtration on nitrocellulose membranes. Deletion of the N-terminal 17 residues or deletion of the β hairpins (residues 72-89) severely decreased S7 affinity for the rRNA. Truncation of the C-terminal portion (residues 138-178), which includes part of the terminal α-helix, significantly affected S7 binding, whereas a shorter truncation (residues 148-178) only marginally influenced its binding. Severe effects were also observed with several strategic point mutations located throughout the protein, including Q8A and F17G in the N-terminal region, and K35Q, G54S, K113Q, and M115G in loops connecting the α-helices. Our results are consistent with the occurrence of several sites of contact between S7 and the 16S rRNA, in line with its role in the folding of the 3' major domain.

Original languageEnglish (US)
Pages (from-to)1649-1659
Number of pages11
JournalRNA
Volume6
Issue number11
DOIs
StatePublished - Nov 28 2000
Externally publishedYes

    Fingerprint

Keywords

  • 16S rRNA
  • Ribosomal protein S7
  • RNA-protein interactions

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Robert, F., Gagnon, M., Sans, D., Michnick, S., & Brakier-Gingras, L. (2000). Mapping of the RNA recognition site of Escherichia coli ribosomal protein S7. RNA, 6(11), 1649-1659. https://doi.org/10.1017/S1355838200001199